Arrhythmia/Electrophysiology - circ.ahajournals.orgcirc.ahajournals.org/content/117/12/1508.full.pdf · Representative examples of ex ovo extracellular recordings in a wild-type HH

Epicardium-Derived Cells in Development of AnnulusFibrosis and Persistence of Accessory Pathways

Denise P. Kolditz, MD; Maurits C.E.F. Wijffels, MD, PhD; Nico A. Blom, MD, PhD;Arnoud van der Laarse, PhD; Nathan D. Hahurij, MSc; Heleen Lie-Venema, PhD;

Roger R. Markwald, PhD; Robert E. Poelmann, PhD;Martin J. Schalij, MD, PhD; Adriana C. Gittenberger-de Groot, PhD

Background—The developmental mechanisms underlying the persistence of myocardial accessory atrioventricularpathways (APs) that bypass the annulus fibrosis are mainly unknown. In the present study, we investigated the role ofepicardium-derived cells (EPDCs) in annulus fibrosis formation and the occurrence of APs.

Methods and Results—EPDC migration was mechanically inhibited by in ovo microsurgery in quail embryos. In ovoECGs were recorded in wild-type (n�12) and EPDC-inhibited (n�12) hearts at Hamburger-Hamilton (HH) stages 38to 42. Subsequently, in these EPDC-inhibited hearts (n�12) and in additional wild-type hearts (n�45; HH 38–42), exovo extracellular electrograms were recorded. Electrophysiological data were correlated with differentiation markers forcardiomyocytes (MLC2a) and fibroblasts (periostin). In ovo ECGs showed significantly shorter PR intervals inEPDC-inhibited hearts (45�10 ms) than in wild-type hearts (55�8 ms, 95% CI 50 to 60 ms, P�0.030), whereas theQRS durations were significantly longer in EPDC-inhibited hearts (29�14 versus 19�2 ms, 95% CI 18 to 21 ms,P�0.011). Furthermore, ex ovo extracellular electrograms (HH 38–42) displayed base-first ventricular activation in44% (20/45) of wild-type hearts, whereas in all EPDC-inhibited hearts (100%, 12/12), the ventricular base was activatedfirst (P�0.001). Small periostin- and MLC2a-positive APs were found mainly in the posteroseptal region of bothwild-type and EPDC-inhibited hearts. Interestingly, in all (n�10) EPDC-inhibited hearts, additional large periostin-negative and MLC2a-positive APs were found in the right and left lateral free wall coursing through marked isolationdefects in the annulus fibrosis until the last stages of embryonic development.

Conclusions—EPDCs play an important role in annulus fibrosis formation. EPDC outgrowth inhibition may result inmarked defects in the fibrous annulus with persistence of large APs, which results in ventricular preexcitation on ECG.These APs may provide a substrate for postnatally persistent reentrant arrhythmias. (Circulation. 2008;117:1508-1517.)

Key Words: electrophysiology � morphogenesis � conduction � arrhythmia

Accessory atrioventricular (AV) pathway–mediated reen-trant tachycardia (AVRT) is a common arrhythmia in

humans.1 It is well established that these accessory pathways(APs) consist of threads of abnormal cardiac musculature thatcross the fibrofatty AV grooves.2 The AP in itself, however,is an enigma, because the causative mechanisms underlyingthe appearance of these AV continuities remain as intriguingas they are unexplained.

Editorial p 1502Clinical Perspective p 1517

It has long been thought that tissues of the endocardial AVcushions and epicardial AV grooves play a key role in the

development of the electrically inert annulus fibrosis, therebycreating the isolating barrier between the atrial and ventriculartissues necessary for normal sequential activation of the heart.3,4

Recently, it was suggested that bone morphogenetic proteinsignaling5 and periostin-induced AV junctional myocardial re-modeling6–8 also play a critical role in configuration of theisolating annulus.

It is not uncommon for annulus fibrosis formation to beincomplete at birth, and consequently, APs can be found inembryonic wild-type quail hearts at near-hatching stages ofembryonic development despite proper His-Purkinje systemconduction and concurrent annulus fibrosis maturation.8 TheseAPs can persist for some time and provide the anatomic

Received July 12, 2007; accepted December 26, 2007.From the Department of Cardiology (D.P.K., M.C.E.F.W., A.v.d.L., M.J.S.), Department of Anatomy and Embryology (D.P.K., N.D.H., H.L.-V.,

R.E.P., A.C.G.-d.G.), and Department of Pediatric Cardiology (N.A.B., N.D.H.), Leiden University Medical Center, Leiden, Netherlands; and Departmentof Cell Biology and Anatomy (R.R.M.), Medical University of South Carolina, Charleston, SC.

The online-only Data Supplement, consisting of an expanded Methods section, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.726315/DC1.

Correspondence to M.J. Schalij, Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, 2300 RC, Leiden,Netherlands. E-mail [email protected]

© 2008 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.107.726315

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substrate for neonatal AVRTs, which usually resolve spontane-ously during the first year of life.8,9 The reasons that AVRTspersist into childhood or adult life are not fully understood, norhave the cell types or instructive signaling routes responsible fornormal annulus fibrosis formation been elucidated fully.

Epicardium-derived cells (EPDCs) migrating through thedeveloping AV dissociated border may be crucial for properannulus fibrosis formation, because the spatiotemporal ex-pression of procollagen I, a marker for collagen type I synthe-sis, closely resembles the migratory patterns of EPDCs,10–12

whereas abundant expression of periostin at the AV junct-ion appears to be spatiotemporally colocalized with thesecells.7,10 Moreover, periostin recently was found to colocalizeand directly interact with collagen type I in murine skin andheart valves.13

EPDCs originate from the proepicardium, which in aviansinitially develops as an outgrowth of the ventral wall of theintraembryonic splanchnopleural coelomic epithelium thatcovers the sinus venosus.14 After �3 days of incubation(Hamburger-Hamilton15 [HH] stage 16 to 18), the proepicar-dium transforms into a cauliflower-like cluster of vesicles (inavians, generally referred to as the proepicardial organ[PEO]16), which enables proepicardial cells to migrate overthe myocardium to form the pericardium and epicardialmonolayer.14 A population of EPDCs is subsequently gener-ated in the subepicardium that results from epicardium-to-mesenchymal transformation.16,17 Most EPDCs are generatedin the intersegmented grooves,10,17–20 subsequently migratingthrough the continuous AV junctional myocardium to popu-late the endocardium-derived AV cushions.10–12

We hypothesize that EPDCs have an inductive role inannulus fibrosis formation, which suggests that postnatal APsmay persist when EPDC migration is inhibited. In wild-typeand in ovo PEO-outgrowth–inhibited quail embryos at post-septated stages of embryonic development, AV conductionwas studied and correlated with annulus fibrosis morphology.

MethodsExperimental PreparationsAnimal experiments were approved by the Committee on AnimalWelfare of the Leiden University and conducted in compliance withthe “Guide for the Care and Use of Laboratory Animals” (NIHpublication No. 85-23, revised 1996). Fertilized eggs of the Japanesequail (Coturnix coturnix japonica, Leiden University) were incu-bated blunt end up at 37.5°C (80% humidity). Embryos were stagedaccording to the HH criteria.15

Outgrowth of the proepicardium was inhibited by performing inovo microsurgery, as described by Männer.21 In brief, on the thirdday of incubation (HH 15–18), a small piece of eggshell membraneis inserted between the dorsal wall of the heart and the pericardialvilli, cranially anchored in the sinuatrial sulcus and caudally con-strained by the coelomic wall (Figure 1).

In Ovo ECG RecordingsAfter termination of incubation at the desired developmental stages(HH 38–42), a subset of quail eggs (EPDC inhibition n�12;wild-type n�12) were prepared for in ovo ECG recording. TheECGs were recorded digitally (Prucka Engineering Inc, Houston,Tex) on a continuous basis for 10 to 15 minutes in a small,custom-built, shielded incubator (37�0.1°C). In ovo ECGs wereevaluated by 2 independent observers. After completion of ECGrecordings, euthanization by decapitation, and subsequent staging,

the embryonic hearts were isolated and prepared for ex ovo extra-cellular electrogram recordings.

Ex Ovo Extracellular Electrogram RecordingsExtracellular electrograms were recorded at HH 38–42 in 45wild-type and 12 EPDC-inhibited hearts during superfusion withoxygenated Tyrode solution, as described previously.8 Definitions,immunohistochemistry, morphometry, and statistical analysis aredescribed in detail in the online-only Data Supplement.

The authors had full access to the data and take full responsibilityfor its integrity. All authors have read and agree to the manuscript aswritten.

ResultsIn Ovo ECG RecordingsThe heart rate (RR interval) was similar in wild-type (n�12) andEPDC-inhibited (n�12) hearts (RR 246�29 versus 252�39bpm, P�0.648). In 3 (25%) of 12 EPDC-inhibited hearts (groupB2) ECGs showed overt ventricular preexcitation reflected by (1)short nonisoelectric PR intervals, (2) initial slurring (delta wave),and (3) resultant lengthening of the QRS complexes. In theseEPDC-inhibited hearts, the PR interval during sinus rhythm wassignificantly shorter (33�12 versus 55�8 ms, P�0.004),whereas QRS intervals were significantly longer (50�9 versus19�2 ms, P�0.004) than in wild-type hearts. EPDC-inhibitedhearts in group B1 (n�9) demonstrated slightly shortened PRintervals (50�6 versus 55�8 ms, P�0.165) and lengthenedQRS intervals (22�3 versus 19�2 ms, P�0.064) comparedwith wild-type hearts. In EPDC-inhibited hearts, the PR andQRS intervals were negatively correlated (Spearman’s��0.314, P�0.320).

The ECG evaluations of 2 independent observers were inagreement: Probability values of 0.830 (PR), 0.344 (RR), and0.187 (QRS) indicated no significant difference between theobservers. Representative examples of in ovo ECG record-ings in a wild-type HH 40 heart (group A), an EPDC-inhibited HH 40 heart (group B1), and an HH 41 EPDC-

Figure 1. Representative example of mechanical EPDC-inhibition technique in an HH 16 quail embryo. A piece of egg-shell membrane (EM) is placed between the right ventricle (RV)and the PEO. Nile blue staining was used to visualize transpar-ent structures. SAS indicates sinuatrial sulcus; CW, coelomicwall; Dao, dorsal aorta; and A, atria.

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inhibited heart with overt preexcitation (group B2) are shownin Figure 2A, 2B, and 2C, respectively. Tables 1 and 2summarize the general electrophysiological (in ovo and exovo) characteristics of all analyzed quail hearts.

Ex Ovo Extracellular Electrogram RecordingsThe AV interval in EPDC-inhibited hearts was significantlyshorter than that in wild-type hearts (62�12 versus 79�26 ms,P�0.033), whereas the RR interval did not differ (125�35versus 111�17 bpm, P�0.693). In line with previous data,8 atthese late stages of embryonic heart development, the ventricularbase was activated prematurely in a considerable number ofembryonic wild-type hearts (20/45, 44%). In contrast, all EPDC-inhibited hearts (12/12, 100%) showed earliest ventricular acti-vation at the ventricular base. In the majority of hearts withpremature ventricular base activation, the right ventricular base(RVB) was the location of first ventricular activation in both

wild-type hearts (RVB�10/20 [50%] versus left ventricular base[LVB]�7/20 [35%]) and EPDC-inhibited hearts (RVB�9/12[75%] versus LVB�3/12 [25%]).

Interestingly, the interval between ventricular base andventricular apex activation was significantly longer in EPDC-inhibited hearts (12�11 ms) than in wild-type hearts showingpremature ventricular base activation (2�2 ms, P�0.001).Representative examples of ex ovo extracellular recordings ina wild-type HH 40 heart (group C) are shown in Figure 3Aand 3B, and recordings obtained in an EPDC-inhibited HH 40heart (group D) are shown in Figure 3C and 3D.

Morphology of the Annulus FibrosisMacroscopically, EPDC-inhibited embryos and their heartswere consistently smaller than wild-type hearts. Furthermore,various known characteristics of the loss-of-PEO-functionphenotype were observed to occur coincidently in these

Figure 2. A, In ovo ECG recordings in a wild-typeHH 40 heart (group A). B, Recordings in an EPDC-inhibited HH 40 heart (group B1) with a shortenedPR interval. C, Recordings in a HH 41 EPDC-inhibited heart with overt preexcitation (group B2).

Table 1. Electrophysiological (In Ovo ECG) Characteristics of Wild-Type and EPDC-Inhibited Hearts

Group, HH Stage n RR, bpm PR, ms QRS, ms

Group A (wild type) 12 246�29 (210–287)* 55�8 (46–74)†§¶#** 19�2 (17–26)‡§#††

HH 38 3 214�4 (210–217) 50�2 (48–52) 18

HH 40 1 271 46 19

HH 41 4 255�27 (222–287) 55�5 (51–62) 18�1 (17–19)

HH 42 4 254�30 (212–280) 61�10 (50–74) 22�3 (20–26)

Group B (EPDC inhibition) 12 252�39 (212–333)* 45�10 (20–57)†� 29�14 (15–60)‡�

Group B1 9 257�43 (213–333) 50�6 (41–57)¶ 22�3 (15–25)#

HH 38 2 213 52�1 (51–52) 21�4 (18–23)

HH 39 2 231�1 (230–232) 51�9 (44–57) 20�7 (15–25)

HH 40 2 308�35 (283–333) 44�4 (41–46) 23�3 (21–25)

HH 41 2 263�46 (231–296) 50�6 (46–54) 22

HH 42 1 282 55 25

Group B2 (overt preexcitation) 3 238�23 (212–257) 33�12 (20–40)#** 50�9 (42–60)††

HH 40 1 256 39 42

HH 41 2 229�24 (212–246) 30�14 (20–40) 54�9 (47–60)

*P�0.648 (Student t test); †P�0.030 (Mann–Whitney U test); ‡P�0.011 (Mann–Whitney U test); §Pearson’s r�0.790, P�0.002;�Spearman’s ��0.314, P�0.320; ¶P�0.165 (Mann–Whitney U test); #P�0.064 (Mann–Whitney U test); **P�0.004 (Mann–Whitney U test); ††P�0.004 (Mann–Whitney U test).

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hearts (Table 3), ie, double-outlet right ventricle with ven-tricular septal defects (2/10), AV valve abnormalities (2/10),great artery abnormalities (1/10), coronary pathology (2/10),and myocardial hypoplasia (2/10).10,12,20–26 The central con-duction axis of the EPDC-inhibited hearts did not show anyhistological abnormalities.

In both wild-type (n�10) and EPDC-inhibited hearts(n�10), small MLC2a-positive APs with comparable vol-umes (1.30�0.40�106 �m3 versus 1.26�0.52�106 �m3,P�0.864) were found primarily in the posteroseptal andmidseptal region of all hearts. In wild-type hearts, additionalsmaller (0.38�0.13�106 �m3) APs were found in the right orleft lateral wall of 6 of 10 hearts (Table 3). Interestingly,however, in all EPDC-inhibited hearts, multiple MLC2a-positive APs in both the right and left lateral free-wallregions with larger volumes than the small lateral APs inwild-type hearts were found (3.14�2.25�106 �m3 versus0.38�0.13�106 �m3, P�0.001; Table 3; Figures 4 and 5).

As expected,8 temporal analysis showed a decrease in APvolume with increasing developmental stage in wild-typehearts (Pearson’s r��0.908, P�0.033). Maturation of theperiostin-positive annulus fibrosis in EPDC-inhibited hearts,however, remained impeded compared with wild-type heartsuntil near-hatching stages of development.

Periostin Expression at the Isolating AV RingPeriostin staining was found at the regions where EPDCs areknown to be present, for example, in the endocardial AV

cushions, in the atrial and ventricular subendocardium, and atvariable expression levels all around the circumference of theAV ring region in both wild-type and EPDC-inhibited hearts. Inboth wild-type and EPDC-inhibited hearts, periostin expressionwas slightly lower in the right than the left AV ring region.

The small, septal, and MLC2a-positive APs in both wild-type and EPDC-inhibited hearts and the small, lateral,MLC2a-positive APs in wild-type hearts stained positive forperiostin. Periostin staining on the annulus fibrosis, however,was interrupted locally at locations where broad lateral APscrossed the annulus in EPDC-inhibited hearts. In Figure 4,representative examples of MLC2a- and periostin-positivestaining in the annulus fibrosis region of wild-type andEPDC-inhibited hearts at HH 39 and HH 41 are given.

Comparison of Immunohistochemical andElectrophysiological DataIn line with our previous report,8 AP location in wild-typepostseptated hearts could not be correlated directly with exovo electrophysiological data; right- or left-sided APs werefound both in hearts that displayed earliest ventricular acti-vation at the RVB or LVB and in hearts with a concurrent orapex-to-base ventricular activation pattern.

In EPDC-inhibited hearts (n�10), however, right-sidedAPs corresponded with premature activation of the RVB, andleft-sided APs corresponded with premature activation of theLVB in 8 of 10 cases. In cases of multiple APs, the location

Table 2. Electrophysiological (Ex Ovo Electrogram) Characteristics of Wild-Type and EPDC-Inhibited Hearts

Group, HH Stage n HR, bpm AV Interval, ms Premature Ventricular Base Activation (n)

Group C (wild type) 45 111�17 (63–120)* 79�26 (41–140)† 20/45 (44%): RVB (10), LVB (7), �(3)

Group C1 (sinus rhythm) 10 82�15 (63–112) 78�15 (63–111)‡ 3/10 (33%): RVB (1), �(2)

HH 38 4 76�12 (63–92) 71�9 (62–81) 0/4 (0%)

HH 39 3 94�17 (77–112) 72�9 (61–78) 2/3 (67%): �(2)

HH 40 3 77�13 (63–90) 94�18 (78–114) 1/3 (33%): RVB (1)

Group C2 (� rhythm) 35 120 79�28 (61–114)‡ 17/35 (49%): RVB (9), LVB (7), �(1)

HH 38 3 120 93�46 (42–132) 3/3 (100%): RVB (2), LVB (1)

HH 39 16 120 72�24 (47–140) 11/16 (69%): RVB (6), LVB (4), �(1)

HH 40 6 120 67�21 (41–89) 1/6 (17%): LVB (1)

HH 41 5 120 96�33 (57–140) 0/5

HH 42 5 120 92�25 (65–127) 2/5 (40%): RVB (1), LVB (1)

Group D (EPDC inhibition) 12 125�35 (65–200)* 62�12 (44–88)† 12 (100%): RVB (9), LVB (3)

Group D1 (sinus rhythm) 6 130�51 (65–200) 68�12 (50–88)§ 6 (100%): RVB (5), LVB (1)

HH 38 1 181 66 1/1 (100%): RVB (1)

HH 39 1 200 67 1/1 (100%): RVB (1)

HH 40 1 65 69 1/1 (100%): RVB (1)

HH 41 2 110�9 (103–116) 59�12 (50–67) 2/2 (100%): RVB (1), LVB (1)

HH 42 1 112 88 1/1 (100%): RVB (1)

Group D2 (� rhythm) 6 120 56�10 (44–68)§ 6 (100%): RVB (4), LVB (2)

HH 38 1 120 68 1/1 (100%): RVB (1)

HH 39 1 120 48 1/1 (100%): LVB (1)

HH 40 2 120 63�3 (61–65) 2/2 (100%): RVB (1), LVB (1)

HH 41 2 120 48�5 (44–51) 2/2 (100%): RVB (2)

Equals sign (�) indicates equal activation of RVB and LVB; � rhythm, paced rhythm.*P�0.693 (Mann–Whitney U test), †P�0.033 (Student t test); ‡P�0.938 (Student t test); §P�0.097 (Student t test).




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of earliest ventricular activation was found to correlate withthe morphologically broadest AP present (Table 3). More-over, conduction velocity along the AP (PR interval) showednegative correlation with the AP volume in EPDC-inhibitedhearts (Pearson’s r��0.696, P�0.037). The observed addi-tional structural defects in the EPDC-inhibited hearts did notcorrelate with the degree of ventricular preexcitation (in ovoPR interval; Spearman’s ��0.000). Interestingly, hearts ingroup B2 demonstrated none to only mild additional structuralabnormalities (Table 3).

DiscussionThe key finding of the present study is that EPDCs areessential for normal annulus fibrosis formation. Conse-quently, inhibition of EPDC migration during cardiogenesismay result in marked defects in the isolating annulus fibrosiswith persistence of broad accessory myocardial AV connec-tions, which results in ventricular preexcitation.

Embryonic Development of the Isolating AnnulusFibrosis: The Role of EPDCs at the AV JunctionDuring development of the electrically inert annulus fibrosis,the primitive slow-conducting continuous AV junctional

myocardium of the looped embryonic heart makes way forconduction through the AV node/His-Purkinje system, whicheventually constitutes the sole AV-conducting pathway of theadult heart.3,27 It is well established that AV junctionalmyocardium is incorporated within the atrial myocardium byfusion of the endocardial AV cushions and the epicardial AVsulcus,3,4 although state-of-the-art studies in the literaturepostulate additional roles for bone morphogenetic proteinsignaling and periostin in annulus fibrosis formation.5–8

Interestingly, expression of periostin mRNA increases signif-icantly in response to mechanically regulated bone morpho-genetic protein signaling in mesenchymal cells in culture.28

Although the contribution of multipotent EPDCs to the heart,both as interstitial fibroblasts (as smooth muscle cells andfibroblasts of the coronary arteries and as mesenchymal cells inthe developing AV cushions) and in their role in formation of thecompact and trabecular myocardium and in Purkinje fiberdifferentiation, has previously been shown to be indispens-able,10,23–25,29–31 the role of EPDCs in annulus fibrosis develop-ment has not been studied in detail until now. The present datashow that in EPDC-inhibited hearts at late postseptated stages ofdevelopment (HH 38–42), large APs coursing through defects

Figure 3. A, Ex ovo extracellular electrograms recorded in a wild-type HH 40 heart (group C) with premature LVB activation (AV interval78 ms). Left atrial (LA) interval (pacing artifact to LA activation) is 20 ms. B, Magnification showing LVB activation 6 ms before left ven-tricular apex (LVA) activation. C, Recordings in an EPDC-inhibited HH 40 heart (group D) demonstrating premature LVB activation (AVinterval 67 ms). D, Magnification showing LVB activation 15 ms before LVA activation.




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in the annulus fibrosis can be found in the right and left lateralfree-wall region, although as previously described in wild-typepostseptated quail hearts,8 additional small APs can be found,mainly in the posteroseptal regions.

After mechanical EPDC inhibition (the present study),epicardial outgrowth is delayed by the insertion of a piece ofeggshell membrane to block the normal cell transfer.11,20,21

Ultimately, regenerating PEO cells growing around the egg-shell membrane together with pericardial mesothelial cellsoriginating from the pharyngeal arch area of the heart form acompensatory epicardium and partially rescue the normalphenotype, thereby yielding embryos with a delayed forma-tion of PEO-derived tissues.12,16,22,23,32 Normally, migrationof subepicardial EPDCs through the continuous AV junc-tional myocardium to the endocardial cushions of the4-chambered heart starts from HH 32 onward.10–12,17–19 Theimpeded development of the AV sulcus in EPDC-inhibitedhearts with consequent persistence of functional large APs atlate postseptated developmental stages (HH 38–42) thusstrongly underscores the importance of EPDCs for normaldevelopment of the annulus fibrosis. A schematic overview of

the proposed role of EPDCs in normal annulus fibrosisformation is depicted in Figure 6.

Periostin Expression in the Developing AnnulusFibrosis of Wild-Type Versus EPDC-InhibitedPostseptated Hearts: Relevance for EPDCFunctioning at the AV JunctionPeriostin is a profibrogenic extracellular matrix protein secretedduring cushion mesenchyme formation and is strongly expressedin collagen-rich fibrous connective tissues that are subjected toconstant mechanical stress in vivo.6,7,33–39 It is thought that thisfasciclin-I–related protein is an inhibitor of the myocardialphenotype under both physiological and pathological condi-tions.8,31,34–36,40,41 Moreover, periostin is likely also essential inmaintaining the integrity of the fibrous heart skeleton of themature heart.36 Multiple cellular mechanisms regulated by peri-ostin might support destabilization of the cardiomyocyte pheno-type and the formation and maintainability of the fibrousscaffold, because periostin is known to bind to fibronectin,tenascin-C, collagen V, and periostin itself.42

The spatiotemporal colocalization of periostin and EPDCsat the AV junction of the developing heart substantiates its

Table 3. Structural Abnormalities, Locations of APs, Cumulative/Individual AP Volumes, and Ventricular Activation Sequences inWild-Type and EPDC-Inhibited Hearts

Embryo #HH

StageStructural

Abnormalities AP LocationCumulative (and Individual) AP

Volumes in 106 �m3Ventricular

Activation Pattern

Wild-type (group A, n�10)† 1.67�0.69*

1 38 S�LL�RL 2.44 (1.46�0.56�0.43) LVA first

2 38 S�LL�RL 2.15 (1.29�0.47�0.39) LVA first

3 39 S�LL�RL 2.79 (2.10�0.43�0.26) LVA first

4 39 S�LL 1.76 (1.33�0.43) LVB first

5 39 S�LL�RL 1.84 (1.50�0.17�0.17) LVB first

6 40 S 1.50 LVA first

7 40 S�RL 1.72 (1.24�0.47) LVA first



10 42 S 1.07 RVB first

EPDC inhibited (group B, n�10)† 6.90�3.26*

Group B1

11 38 DORV�VSD�CA S�LL�RL 10.85 (1.29�4.12�5.45) RVB first

12 38 S�LL�RL 3.22 (0.77�1.29�1.16) RVB first

13 38 S�LL 4.33 (1.97�2.36) LVB first

14 39 AVVA�GAA S�LL�RL 8.19 (1.89�2.49�3.82) LVB first

15 39 DORV�VSD S�LL�RL 7.20 (1.29�0.3�5.62) RVB first

16 40 MH�CA S�LL�RL 6.13 (0.82�0.56�4.76) RVB first

17 41 MH S�LL 2.36 (0.82�1.54) LVB first

Group B2

18 40 S�LL�RL 6.43 (0.52�1.12�4.80) RVB first

19 41 AVVA S�LL�RL 12.95 (1.42�2.32�9.22) RVB first

20 41 S�LL�RL 7.33 (1.80�2.57�2.96) RVB first

S indicates septal; LL, left lateral; RL, right lateral; LVA, left ventricular apex; DORV, double-outlet right ventricle; VSD, ventricular septal defect; CA, coronaryabnormalities; AVVA, AV valve abnormalities; GAA, great artery abnormalities; and MH, myocardial hypoplasia.

Individual volumes are given parenthetically. Bold type indicates largest AP.*Volume measurements according to the Cavalieri method.26

†P�0.001 (Student t test).




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importance for EPDC functioning.7,10 Interestingly, periostinwas recently found to interact directly with collagen type I,13

whereas the migratory patterns of EPDCs in the AV sulcuswere previously established to resemble the spatiotemporalexpression of procollagen I, a marker for collagen type Isynthesis.10–12 The presence of periostin mRNA in the com-pletely epicardium-derived subepicardial mesenchyme fur-ther denotes the EPDC as an important player in the dynamicinterplay between molecular cues and biomechanical deter-minants in the AV junctional myocardium.6,7,10,11

We recently postulated that periostin expression in persis-tent small and mostly posteroseptal-located myocardial APsin wild-type postseptated quail hearts indicates their ultimateperinatal fate as fibrous tissue of the annulus fibrosis.8 In the

present study, periostin expression in the annulus fibrosisregion of EPDC-inhibited postseptated quail hearts was foundto be locally interrupted at sites where large myocardial APsin the lateral free-wall region crossed the isolating annulus,which further substantiates the importance of periostin inEPDC functioning and annulus fibrosis formation. Interest-ingly, similar annulus fibrosis malformations occur in theperiostin-knockout mouse and in mice with a conditionaldeletion of the Alk3 gene and consequent downregulation ofperiostin in the AV region.5,31,43,44

As shown in Figure 4, in both wild-type and EPDC-inhibitedhearts, periostin expression was found in the endocardial AVcushions, 1 of the regions where EPDCs are normally known tobe present.10–12 Expression of periostin in the EPDC-deprived

Figure 4. A, Small MLC2a-positive right posteroseptal AP in an HH 39 wild-type heart. Bar�300 �m. B, Magnification of boxed area.Bar�50 �m. C, Periostin staining. Bar�50 �m. D, Periostin staining (blue) from adjacent section, superimposed on the MLC2a-stained sec-tion, showing marked periostin expression in the myocardial AP. Bar�50 �m. E, Right annulus fibrosis region of an HH 39 wild-type heart.Bar�300 �m. F, Magnification showing complete AV isolation. Bar�2 �m. G, Periostin positivity of the fibrous annulus. Bar�2 �m. H,Periostin-staining superimposed on MLC2a staining. Bar�2 �m. I, Right annulus fibrosis region of an HH 39 EPDC-inhibited heart. Bar�300�m. J, Magnification showing a broad, persistent MLC2a-positive AP in the right anterolateral AV ring region. Bar�1 �m. K, Periostin stain-ing. Bar�1 �m. L, Periostin staining superimposed on MLC2a staining, showing periostin negativity of the myocardial AP. Bar�1 �m. M, Leftannulus fibrosis region of an HH 41 wild-type heart. Bar�300 �m. N, Magnification showing complete AV isolation. Bar�1 �m. O, Periostinpositivity of the fibrous annulus. Bar�1 �m. P, Periostin staining superimposed on MLC2a staining. Bar�1 �m. Q, Left annulus fibrosisregion of an HH 41 EPDC-inhibited heart. Bar�300 �m. R, Magnification showing a broad, persistent MLC2a-positive AP in the left lateral AVring region. Bar�1 �m. S, Periostin staining. Bar�1 �m. T, Periostin staining superimposed on MLC2a staining, showing periostin negativityof the broad lateral AP. Bar�1 �m. RA indicates right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; S, sulcus; Cu, cushion;TV, tricuspid valve; MV, mitral valve; IVS, interventricular septum; and Ao, aorta.




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endocardial AV cushions of the EPDC-inhibited heart, however,indicates that periostin expression is not dependent solely on thephysical presence of EPDCs. Periostin expression can thereforebe speculated to underlie the renowned3,4 but still largelyunknown role of the endocardial AV cushions in annulusfibrosis development, whereas the constant mechanical stress atthe developing AV junction can also be postulated to induceexpression of this profibrogenic protein.33

Annulus Fibrosis Development and FunctionalAP PersistenceNormal formation of the epicardium is described to proceedfrom the point of attachment of the sinoventricular mesocar-dium (the dorsal atrial wall facing the sinus venosus) first tothe dorsal parts of the atrial and the ventricular wall andsubsequently to the ventral wall of the heart.14,17,21 By the end

of HH 26, the myocardium of the looped embryonic heart iscompletely covered by epicardium,14,16 whereas cardiac sep-tation and chamber formation have not yet been complet-ed.45,46 Outgrowth of the right ventricular dorsal wall myo-cardium is 1 of the last processes in cardiogenesis andexpands the right ventricular inflow tract, ultimately resultingin an inevitable shift of the right side of the AV canal tobecome positioned above the right ventricle.45,46 Spatiotem-porally, EPDC population of the right posterior annulusfibrosis region can thus only be achieved after EPDC migra-tion through the expanding dorsal right ventricular wall,which denotes temporal postnatal AP persistence in the rightposteroseptal region of embryonic wild-type quail hearts8 asphysiological perinatal remodeling of the AV junction.

Dyssynchrony in the delicate interplay between EPDCsand AV junctional cells, as shown in the EPDC-inhibitedquail model, results in persistence of large lateral APs.Although EPDCs derived from the compensatory epicardiumultimately do arrive at the epicardial AV sulcus of EPDC-inhibited hearts,12,32 these cells possibly encounter AV junc-tional cardiomyocytes already impermissive for EPDC inter-action and thus miss the appropriate time window for theirintended rescue of the normal cardiac phenotype.

Persistent APs in wild-type versus EPDC-inhibited postsep-tated quail hearts also displayed divergent electrophysiologicalcharacteristics. In line with our previous report,8 morphologi-cally persistent APs in the wild-type heart that give rise topremature ventricular activation could be found in a consider-able number of cases. Inhibition of EPDC migration, however,resulted in persistence of large APs with a relatively highconduction velocity (short in ovo PR and ex ovo AV intervals),neither of which was observed in any of the wild-type hearts,and premature ventricular base activation in all cases.

Moreover, overt ventricular preexcitation was observed ina subgroup of in ovo ECGs in EPDC-inhibited hearts.Interestingly, these EPDC-inhibited hearts all demonstratedthe 3 main clinical ECG features of ventricular preexcitationsyndromes47,48: (1) a short PR interval, (2) initial slurring ofthe QRS complex, known as the delta wave, and (3) aresultant prolonged QRS complex (Figure 2).

Clinical SignificanceIn children, the first episodes of AP-mediated AVRT occurbefore birth or in the first months of life in �60% of cases

Figure 5. Schematic figure of mitral (MV) and tricuspid (TV)valve annuli in wild-type (A) and EPDC-inhibited (B) quail heartswith AP locations and corresponding AP volumes. CS indicatescoronary sinus.

Figure 6. Annulus fibrosis formation andthe role of EPDCs. A, Continuous AV junc-tional myocardium in the looped embryonicheart. B, From HH 32 onward, subepicardialEPDCs in the AV sulcus (S) migrate throughthe continuous AV junctional myocardium toultimately populate the endocardial AV cush-ions (Cu). C, In the normal 4-chamberedheart, EPDCs continue populating the AVcushions and favor 2 positions: (1) the myo-cardial/endocardial cushion interface and (2)subendocardially at the luminal face of theAV cushions. D, Impeded development ofthe annulus fibrosis in EPDC-inhibited heartsresults in subsequent persistence of largeAPs in the postseptated heart. A indicatesatrium; V, ventricle; S, sulcus; and Cu,cushion.




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and resolve spontaneously in most cases before the age of 1year, whereas recurrence of tachycardia at the age of 8 to 10years occurs in approximately the remaining 30%.1,9 Bol-stered by the normal postnatal evolutionary process of ana-tomic molding and shaping of the heart to facilitate adjust-ment to the increasing body mass and changes in vascularpressures, we previously postulated8 that under physiologicalcircumstances, persistent functional APs at near-hatchingstages of avian development provide the anatomic substratefor spontaneously resolving neonatal AVRTs.

However, any delay in EPDC migration in the developingheart may result in imperfect annulus fibrosis developmentand consequently in AP persistence. Clinically, most patientswith persistent APs are not affected by additional cardiacpathology, although in some cases of ventricular preexcita-tion syndromes (eg, Wolff-Parkinson-White syndrome), APpersistence coincides with congenital cor vitia (for example,Ebstein’s anomaly).9,49 When EPDC migration is blockeddirectly after PEO formation, as in the present study, multiplemild to severe congenital heart defects will occur.10,12,22–25 Inhumans, genetic alterations that affect EPDC functioningduring development and occur after structural configurationof the heart has been completed may result in postnatal APpersistence in a structurally normal heart.1,47

Study LimitationsAlthough we showed both functionally and morphologicallythat EPDCs are indispensable for proper annulus fibrosis forma-tion, we were unable to demonstrate subsequent AP persistencein hatched or adult EPDC-inhibited quail hearts. Unfortunately,mechanical EPDC inhibition yields embryos in which the degreeof epicardial outgrowth inhibition is directly related to theseverity of the cardiac abnormalities and thus to embryoniclethality.10,22,23,25 Although the operated embryos that survivedbeyond HH 38 were typically those only mildly affected bycomorbidity, EPDC-inhibited quail embryos are relatively smalland appear to lack sufficient reserve to survive postnatally.Interestingly, severe growth retardation, postnatal lethality, anddwarfism in adult life have recently been described in theperiostin-null mouse.43,44

ConclusionsEPDCs appear to be essential for proper formation of theisolating annulus fibrosis. Inhibition of EPDC migrationduring cardiogenesis may result in marked defects in theannulus fibrosis with persistence of broad APs, which func-tionally results in ventricular preexcitation. Although underphysiological conditions, small septal APs in the wild-typeheart temporarily remain functionally active,8 broad lateralAPs in the EPDC-inhibited heart might provide a pathologi-cal substrate for postnatally persistent APs and AVRTs intochildhood or adult life.

DisclosuresNone.

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CLINICAL PERSPECTIVEAtrioventricular reentrant tachycardia is a common arrhythmia in both children and adults; however, the causalmechanisms underlying the appearance of accessory pathways remain a subject of debate. During cardiogenesis, initialslow conduction over the circumferential myocardial AV continuity, which results in sequential activation of the preseptatedheart, is replaced by apex-to-base conduction through the specialized AV node/His-Purkinje system in the septated heart.Concurrently, incorporation of the AV junctional myocardium in the lower atrial rim by fusion of the endocardial AVcushions and epicardial AV sulcus results in formation of the isolating annulus fibrosis. Migration of multipotentepicardium-derived cells (EPDCs) through the continuous AV junctional myocardium, ultimately reaching theendocardium-derived AV cushions, spatiotemporally correlates with annulus fibrosis formation. The AV junction has beenpostulated to be subject to physiological perinatal remodeling, which temporarily leaves functional small accessorypathways as anatomic substrates for spontaneously resolving neonatal AV reentrant tachycardias. Dyssynchrony in thedelicate interplay between EPDCs and AV junctional cells, as shown in the EPDC-inhibited quail model in the presentstudy, may result in marked defects in the isolating annulus fibrosis, with the persistence of large accessory pathwaysfunctionally resulting in ventricular preexcitation. We speculate that absence of EPDCs or a delay in EPDC migrationresults in the persistence of pathological substrates for postnatally persistent accessory pathways and AV reentranttachycardias into childhood or adult life.




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Adriana C. Gittenberger-de GrootHahurij, Heleen Lie-Venema, Roger R. Markwald, Robert E. Poelmann, Martin J. Schalij and Denise P. Kolditz, Maurits C.E.F. Wijffels, Nico A. Blom, Arnoud van der Laarse, Nathan D.

Accessory PathwaysEpicardium-Derived Cells in Development of Annulus Fibrosis and Persistence of

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Arrhythmia/Electrophysiology - circ.ahajournals.orgcirc.ahajournals.org/content/117/12/1508.full.pdf · Representative examples of ex ovo extracellular recordings in a wild-type HH